Interest in regeneration therapy is growing. Over the past few years, several studies have been conducted on the generation of hepatocytes using bone marrow and liver stem-like cells obtained from living bodies [Schwartz, R. E., Reyer, M., Koodie, L., Jiang, Y., Blackstad, M., Lund, T., Lenvik, T., Johnson, S., Hu, W. S., Verfaillie, C. M., “Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.”, J. Clin. Invest. 109, 1291-1302, 2002; and Suzuki, A., Zheng, Y. W., Kaneko, S., Onodera, M., Fukao, K., Nakauchi, H., Taniguchi, H., “Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.”, J. Cell Biol. 156: 173-184, 2002]. At the same time, the present inventers observed that embryonic stem (ES) cells can differentiate. ES cells were first established by cloning cell lines that comprised several differentiation activities, using the inner cell masses of mouse blastocysts on dishes pretreated with gelatin and comprising misogynic C-inactivated STO fibroblasts [Evans, M. J., Kaufman, M. H., “Establishment in culture of pluripotential cells from mouse embryos.”, Nature 292:154-156, 1981; Martin, G. R., “Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.”, Proc. Natal. Aced. Sic. USA. 78: 7634-7638, 1981; and Bradley, A., Evans, M., Kaufman, M. H., Robertson, E., “Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.”, Nature. 309: 255-256, 1984]. When in the presence of a feeder cell layer or leukemia inhibitory factor (LIF), ES cells multiply semi-permanently under conditions that maintain their undifferentiated state [Williams, R. L., Hilton, D. J., Pease, S. et al, “Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.”, Nature 336: 684-687, 1988]. When allowed to differentiate in a suspension culture, ES cells form spherical multi-cellular aggregates, called embryonic bodies (EBs). EBs have been shown to comprise a variety of cell populations. The processes of neuron, cardiac muscle and hematopoietic cell differentiation have been investigated using ES in vitro differentiation systems [Schmitt, R. M., Bruyns, E., and Snodgrass, H., “Hematopoietic development of embryonic stem cells in vitro, cytokine and receptor gene expression.”, Genes Dev. 5: 728-740, 1991; Keller, G. M., “In vitro differentiation of embryonic stem cells.”, Cur. Open. Cell Boil. 7: 862-869, 1995; Sanchez-Carpintero, R., and Narbona, J., “Executive system: a conceptual review and its study in children with attention deficit hyperactivity disorder.”, Rev. Neurol. 33: 47-53, 2001; and Bain, G., Kitchens, D., Yao, M., Huettner, J. E., and Gottlieb, D. I., “Embryonic stem cells express neuronal properties in vitro.”, Dev. Boil. 196: 342-357: 1995]. Thus, ES cell differentiation provides a valuable model for the study of visceral endoderm formation, and provides new possibilities for transplantation medicine.
Recently, biomaterials were used in the cell therapy of a number of daisies patients. For example, by culturing for four months in a medium supplemented with fibroblast growth factor (FGF)-2, ES cells transfected with hepatocyte nuclear factor (HNF)-3β were differentiated from albumin-induced cells [Ishizaka, S., Shiroi, A. et al., “Development of hepatocytes from ES cells after transfection with the HNF-3β gene.”, FEBS J. 16: 1444-1446, 2002]. After 18 days of culture, EBs differentiated into hepatocytes, and were plated onto gelatin-coated dishes and incubated for 21 to 30 days without LIF and growth factors [Abe, K., Niwa, H., Iwase, K., Takiguchi, M., Mori, M., Abe, S., and Abe, K., “Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies.”, Exp. Cell Res. 229: 27-34. 1996; and Miyashita, H., Suzuki, A., Fukao, K., Nakauchi, H., and Taniguchi, H., “Evidence for hepatocyte differentiation from embryonic stem cells in vitro.”, Cell Transplantation. 11: 429-434, 2002]. The product of the EB-derived hepatocytes was plated on collagen type I-coated dishes and cultured for 18 days along with growth factors (acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF), and oncostatin M (OsM)), dexamethasone, and IST (a mixture of insulin and transferin). However, in vitro EB formation was necessary in all cases where hepatocytes were formed from ES cells. Functional cells produced from EBs encounter several problems, such as teratoma formation. In addition, the formation of EBs from ES cells is laborious, and the differentiation rate is generally low. Differentiation to a number of other cells often occurs, thus calling for a hepatocyte purification Step. Few studies have attempted to differentiate cells from ES cells without mediating EBs. The only example of such a study is that in which Aubert et al induced nerve cell differentiation (Aubert, J., Dunstan, H., Chambers, I., and Smith, A., “Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation.”, 20: 1240-1245, 2002).
Mesenchymal stem cells (MSCs) were first isolated from bone marrow by Friedenstein in 1982 by simple plating on plastic in the presence of fetal calf serum (FCS) (Pittenger M. F. et al., Science 284, 143-147, 1999). Human MSCs isolated from bone marrow (BM) aspirates share a general immunophenotype, and are uniformly positive for SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a and CD124, but negative for CD14, CD34, and the leukocyte common antigen CD45 (Pittenger F. M. et al., supra, 1999). In addition, the expression of VCAM-1, LFA-3, and HLA MHC Class I molecules in human MSCs was shown by flow cytometry analysis, suggesting the ability of these cells to undergo appropriate interaction with T-cells.
Human MSCs are multipotent, and they can differentiate into at least three lineages (osteogenic, chondrogenic, and adipogenic) when cultured under defined in vitro conditions (Pittenger F. M. et al., supra, 1999). Previously attempts at differentiation of mature hepatocytes from adult BM including human MSCs (CD34-positive cell fraction) have been reported (Camper S. A. and Tilghman S. M., Biotechnology 16, 81-87, 1991; Nahon J. L,. Biochimie. 69, 445-459, 1987; and Medvinsky A. and Smith A., Nature 422, 823-825, 2003). However, there are no reports of the induction of functional hepatocytes by direct differentiation in vitro.